Glucocorticoid induces GSDMD-dependent pyrolysis in PC12 cells via endoplasmic reticulum stress

Objective The present study explored whether pyroptosis is involved in the injury process of PC12 cells induced by glucocorticoid (GC) and the regulatory relationship between endoplasmic reticulum stress (ERS) and pyrolysis. Methods LDH leakage of PC12 cells was detected by LDH assay. The number of dead cells was detected by SYTOX green nucleic acid staining. The levels of IL-1β and IL-18 in the supernatants was detected by ELSIA assay. The expression levels of glucose regulated protein 78 (GRP78), cleaved gasdermin D-NT (cleaved-GSDMD-NT), NLR-pyrin domain-containing 3 (NLRP3) and cleaved-caspase-1 were observed by immunofluorescence staining and western blot. Results The LDH assay revealed that GC exposure significantly increased the release of LDH. The results of SYTOX green acid staining showed that GC exposure significantly increased the number of SYTOX green acid-positive cells. The ELSIA assay revealed that GC exposure significantly increased the levels of IL-1β and IL-18 in the supernatants. The results of immunofluorescence staining and western blot showed that GC exposure significantly increased the expression of GRP78, cleaved-GSDMD-NT, NLRP3 and cleaved caspase-1. Treatment with the ERS inhibitor tauroursodeoxycholate (TUDCA) and siRNA GSDMD attenuated related damage and downregulated the expression of the abovementioned proteins. Conclusion The present study clearly demonstrated that GC exposure can induce GSDMD-dependent pyrolysis, and ERS is involved in the above damage process.


Results
The LDH assay revealed that GC exposure significantly increased the release of LDH. The results of SYTOX green acid staining showed that GC exposure significantly increased the number of SYTOX green acid-positive cells. The ELSIA assay revealed that GC exposure significantly increased the levels of IL-1β and IL-18 in the supernatants. The results of immunofluorescence staining and western blot showed that GC exposure significantly increased the expression of GRP78, cleaved-GSDMD-NT, NLRP3 and cleaved caspase-1. Treatment with the ERS inhibitor tauroursodeoxycholate (TUDCA) and siRNA GSDMD attenuated related damage and downregulated the expression of the abovementioned proteins.

Introduction
As an important steroid hormone, GC is secreted by the adrenal cortex tract [1]. Physiological doses of GC can regulate material metabolism and maintain the body's life activities [2]. When the body is stimulated by various internal and external factors, the hypothalamus pituitary adrenal (HPA) axis is activated, which can increase the synthesis and secretion of GC and produce anti-inflammatory, anti-infective, anti-shock, and immunosuppressive effects to maintain homeostasis [3]. Previous studies have shown that long-term high-intensity stress can induce the body to secrete a large amount of GC [4], and high-concentration GC can penetrate the blood-brain barrier, causing neuronal damage or even death and multiple mental disorders [5]. Cell models are efficient ways to identify the underlying mechanism of alcohol-induced neuronal apoptosis. As a cell line derived from rat adrenal pheochromocytoma, differentiated PC12 cells exhibit the general characteristics of neuroendocrine cells and are widely used in neurophysiology and neuropharmacology research. Our previous study has showed that highconcentration GC exposure can significantly inhibit the viability of PC12 cells and induce cell apoptosis. However, little is known regarding the mechanism of damage induced by GC. The endoplasmic reticulum (ER) is an important organelle mainly responsible for protein synthesis, folding, and transport [6]. The correct and efficient folding of proteins plays an important role in cell survival and physiological functions [7]. Disruption of ER homeostasis can cause an accumulation of unfolded or misfolded proteins within the ER, which can activate ERS [8,9]. However, persistent ERS can switch the cytoprotective function of the unfolded protein response (UPR) into cell death-promoting mechanisms [10]. There are three unique stress sensors located at the ER membrane: inositol-requiring protein 1α (IRE1α), PKR-like eukaryotic initiation factor 2α kinase (PERK), and activating transcription factor 6 (ATF6) [11]. Under normal physiological conditions, the above three transmembrane proteins all bind to the molecular chaperone GRP78 in an inactive state [12]. When ERS occurs, the accumulated unfolded or misfolded proteins competitively bind and sequester GRP78 away from PERK, IRE1, and ATF6. This leads to the activation of these three transducers, which in turn triggers a series of molecular reactions that cause various pathological damages, such as inflammation, apoptosis, and autophagy [13,14]. Our previous study showed that the PER-K-ATF4-CHOP and IRE1-ASK1-JNK pathways were involved in the GC-induced injury process of PC12 cells.
Pyroptosis is a type of programmed cell death [15]. As the executive protein of pyroptosis, gasdermins (GSDMs) are a recently characterized protein family encoded by six paralogous genes: gasdermin A (GSDMA), gasdermin B (GSDMB), gasdermin C (GSDMC), gasdermin D (GSDMD), gasdermin E (GSDME) and pejvakin (PJVK) [16]. GSDMs usually contain two protein functional domains (N-terminal and C-terminal domains), which form a conserved two-domain structure and share an autoinhibitory mechanism [17]. Under pathological conditions, GSDM-NT and GSDM-CT can be separated causing the autoinhibition effect to disappear. Cleaved-GSDM-NT can be connected to phospholipids on the cell membrane, resulting in their polymerization and insertion into the cell membrane to generate hole-like structures with an inner diameter of approximately 10-16 nm. As nonselective pores, GSDM pores are sufficient to allow the inflow of extracellular H 2 O and Ca 2+ to cause cell swelling and disintegration; subsequently, the cell contents flow out, ultimately resulting in the release of IL-1β and IL-18 and pyroptosis [18].
However, whether pyroptosis is involved in the injury process of PC12 cells induced by glucocorticoid remains unclear as does the regulatory relationship between ERS and pyrolysis. For the present study, we aimed to investigate the mechanism of cell injury induced by GC at the cellular level and provide morphological evidence.

Cell culture and treatment
Cell culture and treatment were executed as described previously [19]. PC12 cells were cultured in RPMI 1640 medium supplemented with 7.5% fetal bovine serum, 5% horse serum, 100 U/mL penicillin/streptomycin, and 110 μg/mL sodium pyruvate solution at 37˚C in a humidified atmosphere of 5% CO2. PC12 cells were incubated with 50 ng/mL NGF 2.5S for 48 h to induce neurite formation and imitate neurons. After seeding in appropriate petri dishes, differentiated PC12 cells were used for subsequent experiments.
For experiments involving siRNA transfections, PC12 cells were plated in 6-well plates to reach a confluence of 50%~60%. Then transfections were fulfilled using 5μl Lipofectamine 2000™ added to 250μl of Opti-MEM medium, which was incubated at room temperature for 5 min. In another tube, 5μl of 50nM siRNAs were added into 250μl Opti-MEM medium before both tubes were mixed and incubated at room temperature for 20 min. Cells were incubated in this mixture for 8 hours.

LDH release assay
Briefly, PC12 cells were treated with 100 μM DEX for 24 h. Then, supernatants were extracted and treated with an LDH assay kit. Finally, treated samples were detected at 450 nm by Multiskan Go (Waltham, MA, USA).

SYTOX green nucleic acid staining
PC12 cells were treated with 100 μM DEX for 24 h. Cells were washed three times in Hank's balanced salt solution (HBSS) and incubated with SYTOX green acid staining solution for 20 min in the dark. Then, the cells were washed three times in HBSS and images were acquired by a confocal laser scanning microscope (Leica, Ernst-Leitz-Strasse, Wetzlar, Germany).

ELISA assay
Briefly, PC12 cells were treated with 100 μM DEX for 24 h. Then, supernatants were extracted and treated according to the ELISA kit. Finally, treated samples were detected at 450 nm by Multiskan Go (Waltham, MA, USA).
In the present study, Image-Pro Plus 5.1 (Media Cybernetics, Houston, TX, USA) was used to count the number of cells and detect the fluorescence intensity (IOD) of all cells. The average IOD was used to illustrate the expression levels of the corresponding proteins.

Western blot
Total protein (50 μg of protein/lane) was put into SDS-PAGE gels, then detached by electrophoresis and delivered to PVDF membranes. Subsequently, the target areas of the membranes were hatched with rabbit antibodies for GRP78 (1:1000), cleaved GSDMD-NT (1:1000) and βactin (1:2000) overnight at 4˚C. Then, the target areas of the membranes were hatched with goat anti-rabbit IgG (H+L) secondary antibody (#31210, Invitrogen, CA, USA) and detected by X-ray film with an enhanced chemiluminescence system. ImageJ 1.48 (National Institutes of Health, Maryland, USA) was applied to investigate the intensity of the bands.

Statistical methods
The results are presented as mean ± SEM. Data were analyzed by one-way ANOVA followed by a post hoc least significant difference (LSD) t-test to determine specific group differences, and the effects of TUDCA and siRNA GSDMD were analyzed by two-way ANOVA. All statistical analyses were performed using SPSS 21.0. Significance was defined as P < 0.05 for all statistical tests.

LDH leakage of PC12 cells
Compared to the control group (11.60 ± 1.08), the LDH leakage of PC12 cells remained at a low level in the TUDCA group (10.40 ± 1.91, P > 0.05) and was significantly increased in the DEX group (38.20 ± 2.31, P < 0.01) and DEX + TUDCA group (22.20 ± 1.39, P < 0.01). Compared to the DEX group, the LDH leakage of PC12 cells was significantly decreased in the DEX + TUDCA group (P < 0.01) (Fig 1A).

The number of SYTOX positive cells
Compared to the control group (3.6 ± 0.81), the number of SYTOX positive cells remained at a low level in the TUDCA group (4.0 ± 0.71, P > 0.05) and was significantly increased in the DEX group (18.20 ± 0.86, P < 0.01) and DEX + TUDCA group (11.20 ± 0.87, P < 0.01). Compared to the DEX group, the number of SYTOX positive cells was significantly decreased in the DEX + TUDCA group (P < 0.01) (Fig 1B).
Compared to the siRNA NC group (4.7 ± 0.70), the number of SYTOX positive cells remained at a low level in the siRNA GSDMD group (5.6 ± 0.83, P > 0.05) and was significantly increased in the DEX + siRNA NC group (22.2 ± 1.67, P < 0.01) and DEX + siRNA GSDMD group (12.6 ± 0.82, P < 0.01). Compared to the DEX + siRNA NC group, the number of SYTOX positive cells was significantly decreased in the DEX + siRNA GSDMD group (P < 0.01) (Fig 1D).

The levels of IL-1β and IL-18 in the supernatants
Compared to the control group (216.20 ± 23.15), the level of IL-1β remained at a low level in the TUDCA group (241.80 ± 31.62, P > 0.05) and was significantly increased in the DEX

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Compared to the DEX group, the expression of GRP78 was significantly decreased in the DEX + TUDCA group (P < 0.01) (Fig 4).

GRP78 and cleaved GSDMD-NT protein expression in PC12 cells by western blot
Compared to the control group (0.40 ± 0.04), the expression of GRP78 remained at a low level in the TUDCA group (0.46 ± 0.02, P > 0.05) and was significantly increased in the DEX group (0.85 ± 0.07, P < 0.01) and DEX + TUDCA group (0.62 ± 0.04, P < 0.05). Compared to the DEX group, the expression of GRP78 was significantly decreased in the DEX + TUDCA group (P < 0.05) (Fig 8A).

Discussion
The previous study showed that PC12 cells have obvious characteristics of neurons after being treated with NGF and abundantly express glucocorticoid receptors [19], which indicates that PC12 cells are suitable for investigating the mechanism of GC-induced neuronal injury. The physiological dose of GC can regulate material metabolism and has anti-inflammatory, antiinfective, anti-shock, and immunosuppressive effects to maintain homeostasis [3]. However, research indicates that continuous stress causes excessive secretion of GC, which can infiltrate the blood-brain barrier and induce neuronal injury [5]. Our previous study showed that the viability of PC12 cells is significantly reduced and the rate of apoptosis is significantly increased after treatment with 100 μM DEX for 24 h [19]. In the present study, the results of LDH assays showed that GC exposure can significantly increase the release of LDH. The results of SYTOX green acid staining showed that GC exposure can significantly increase the number of SYTOX green acid-positive cells. Also, the results of ELISA assay showed that GC

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exposure can significantly increase the levels of IL-1β and IL-18 in the supernatants. The above results showed that GC can significantly damage the cell membrane and increase the leakage of cell contents, indicating that pyrolysis may be involved in the process of PC12 cell damage induced by GC.
Pyroptosis, also known as cell inflammatory necrosis, is a kind of programmed cell death [20]. Among the six protein subtypes of pyrolysis, the GSDMD-related classical pyrolysis pathway has been fully studied [17]. As a member of the nucleotide-binding domain like receptor (NLR) family, NLRP3 can activate the inflammasome and induce pyroptosis [21]. The NLRP3 inflammasome is composed of NLRP3, apoptosis associated speck-like protein containing a CARD (ASC), and proinflammatory caspase-1 [22]. When cells are subjected to various internal and external stimuli, NLRP3 oligomerizes and exposes pyrin domains (PYDs), which can interact with ASCs. Then, the CARDs of ASCs interact with pro-caspase-1, which can enable caspase-1 activation [23]. Cleavage of GSDMD is achieved by caspase family proteins. Caspase-1 and caspase-11 (caspase-4/5 in humans) can recognize and cleave GSDMD to form cleaved GSDMD-NT, which can induce pyrolysis [24]. In the present study, the expression levels of NLRP3, cleaved caspase-1 and cleaved GSDMD-NT were significantly increased after treatment with DEX, while after treating PC12 cells with siRNA GSDMD, the expression of cleaved GSDMD-NT was significantly decreased, the release of LDH and the number of SYTOX green acid-positive cells were also significantly reduced, which indicating that the NLRP3/caspase-1/GSDMD pathway was involved in pyrolysis of PC12 cells induced by GC.
As a widely distributed membranous organelle, the ER is responsible for the synthesis, folding, and transport of most proteins in eukaryotic cells [25]. Unfolded or misfolded proteins can lead to the accumulation of abnormal proteins in the cell, which then activates the UPR and leads to ERS [26]. Under ERS conditions, the expression of GRP78 is significantly upregulated, and is often used as an activation marker of ERS [27]. In the present study, the expression of GRP78 was significantly increased after treatment with DEX, indicating that GC can induce ERS in PC12 cells. Application of the ERS inhibitor TUDCA significantly reduced the release of LDH and the number of SYTOX green acid-positive cells, the levels of IL-1β and IL-18 in the supernatants and the expression levels of GRP78, NLRP3, cleaved caspase-1, and cleaved-GSDMD-NT were also significantly decreased. The above results suggest that ERS was involved in the GC-induced pyrolysis of PC12 cells via the NLRP3/caspase-1/GSDMD pathway.
In conclusion, the present study clearly demonstrated that GC exposure can induce GSDMD-dependent pyrolysis, and ERS is involved in the above damage process. We believe these novel findings will provide a new research perspective on the mechanism of GC-induced neuronal injury.